Since its conception more than 27 years ago, the principle of particle counting and sizing invented by Wallace H. Coulter has resulted in numerous methods and apparatuses for the electronic counting, sizing and analysis of microscopic particles, which are scanned in a fluid suspension, as shown by the pioneer U.S. Pat. No. 2,656,508 to Coulter. In this prior art particle analyzer, a D.C. electric current flow is established between two vessels by suspending electrodes in the respective bodies of the suspension fluid. The only fluid connection between the two bodies is through an orifice; hence, an electric current flow and field are established in the orifice. The orifice and the resultant electric field in and around it constitute a sensing zone. As each particle passes through the sensing zone, for the duration of the passage, the impedance of the contents of the sensing zone will change, thereby modulating the current flow and electric field in the sensing zone, and hence causing the generation of a signal to be applied to a detector suitably arranged to respond to such change. (The mark "Coulter" is a registered trademark, Registration No. 995,825, of Coulter Electronics, Inc. of Hialeah, Fla.)
In the commercial apparatus constructed in accordance with the heretofore mentioned U.S. Pat. No. 2,656,508, field excitation has been supplied by a direct current or low frequency source. The electrical change, i.e., D.C. signal, caused by the passage of a particle through the electric field of small dimensions, excited by a direct or low frequency current, is approximately proportional to particle size. A direct current is considered to be of zero frequency in this application. However, the impedance sensing principle has been materially expanded to provide information concerning particles being studied, not limited only to characteristics due to the size of particles, but including characteristics due to the composition and nature of the material constituting the particles, as disclosed in U.S. Pat. No. 3,502,974 to Coulter et al and U.S. Pat. No. 3,502,973 to Coulter et al. These prior art apparatuses generally have at least two current sources, both of which are applied to the sensing zone simultaneously, one having a radio frequency (RF) and the other being the previously described "zero frequency" direct current (DC) or, alternatively, having a sufficiently low frequency that the reactive part of the particle impedance has a neglible effect on the response of the apparatus. One of the useful particle descriptors that can be obtained from this dual source arrangement is the "internal conductivity" or "opacity" of the particles. More specifically, with biological cells, their membranes have a very high resistivity in the range of a dielectric; however, the internal portion of the cell is fairly conductive. The RF current passes through the cell's membrane, thereby generating a detectable RF signal which correlates to the "internal conductance" for each particle. When the D.C. size signal for a cell is divided into the RF signal for that cell, a measurement correlating with the "internal conductivity" of the cell is obtained.
The drawback of the above described particle analyzers is that the size and internal conductivity measurements generally do not correlate exactly with the actual or true volume and internal conductivity, respectively, of the cell. Generally, due to the hydrodynamic focusing in most apparatuses, elongated particles will be aligned with their elongated axis substantially parallel to the center axis of the orifice. With two equal volume particles, one being spherical and one being elongated, the spherical particle, while passing through the orifice, will have a greater cross section perpendicular to the current flow than the elongated particle. Hence, the spherical particle will distort the field in such a manner that it will give a greater measured size signal than the elongated particle, despite their equal volumes. Consequently, particles have been classified as to their shape by a term called "shape factor" which is used to correct their measured D.C. size signal. For instance, if an extremely elongated particle is assigned a shape factor of 1.0, then the spherical particle of the same volume has a shape factor of 1.5.
To correct for the inaccuracies introduced into the measured parameters by the particle's shape, the shape factor can be accurately measured on a cell by cell basis by obtaining a third signal, such as length, in addition to the RF and DC signals and then correcting the measured parameters to obtain accurate values, as described in allowed U.S. patent application No. 096,945, filed Nov. 23, 1979, to Groves et al. Now U.S. Pat. No. 4,298,836. However, this arrangement has the disadvantage of requiring an optical source and detector for obtaining the required length by making a "time of flight" measurement.
A second drawback of the prior art electronic volume sensing particle analyzers is that slit scanning of the individual cell cannot be accomplished, such scanning being possible only with optical particle analyzers, as shown in U.S. Pat. No. 3,657,537 to Wheeless. More specifically, with the optical particle analyzers, a narrow illuminating beam, having a width less than the length of the cell traversing the same, excites fluorescence from a stained cell. In this manner the internal constituents of the cell are examined, such as the relative sizes of the cell's nucleus and cytoplasm. It is known that there are differences in internal resistivities of different portions of the cell, such as, for example, between the nucleus and the surrounding cytoplasm. However, in these prior art analyzers, these internal differences have not been measureable or subject to being quantified, due to the sensing zone created by the electric field being always much longer in length than the cell. For instance, a sensing zone in a 100 micron long aperture will be substantially longer than the 100 microns and will typically receive cells having lengths in the range of 10 microns.
A third drawback of the prior art electronic volume sensing analyzers is that the volume of the sensing zone is much greater than the volume of a cell, resulting in some loss in signal resolution. For instance, the smallest practical orifice size would have a cross-sectional diameter of 50 microns and a length of 50 microns, as compared to the 10 micron diameter of the cell, resulting in a volume 187 times greater than the cell. The ratio of the volumes of the actual sensing zone and the cell is even greater.
A fourth drawback of the electronic volume particle analyzers of the prior art is that in order for microscopic sized particles to be analyzed, the aperture must form a constricted passageway for the electrical current, generally having a diameter no greater than 50 to 100 microns to create sufficient current densities. These microscopic dimensions make difficult the fabrication of the aperture within reasonable tolerances and in operation frequently lead to the aperture becoming clogged with debris.
In U.S. Pat. No. 3,821,644 to Gohde et al. an electrode arrangement is shown for creating a unidirectional electric sensing field between plates for making the detected signals insensitive to particle trajectories. More specifically, each particle is forced to traverse or cut the same amount of current, regardless of the displacement of the particle's trajectory from the center axis of the orifice, due to the field being unidirectional. The width of this sensing field is limited by how thin the center electrodes can be made, which is generally in excess of 100 microns. As taught by this patent, the sensing zone is much longer in length than the cell; hence, the peak amplitude of the signal occurs when the entire cell is within the confines of the sensing zone.
The previously mentioned U.S. Pat. Nos. 3,502,974, 3,502,973 and 4,298,836 are incorporated herein by specific reference thereto.